Pore-forming cytolysins are produced by virtually all genera of pathogenic Gram-positive bacteria. The most widely distributed group is the CDC family of which Streptolysin O (SLO) is a prominent member. SLO is produced by Streptococcus pyogenes, an important cause of pharyngitis and other serious diseases including scarlet fever, rheumatic fever and necrotizing faciitis. However, how SLO acts to promote the pathogenesis of any of these diseases is not well-understood. In this project, we have found that SLO plays a central role in the Cytolysin-Mediated Translocation (CMT) pathway, a novel pathway that acts to translocate the S. pyogenes NAD-glycohydrolase (SPN) across the host cell membrane and into its cytosol. CMT likely makes an important contribution to pathogenesis as cytosolic SPN is cytotoxic for cultured cells. However, how SLO functions to translocate SPN, and how SPN contributes to cytotoxicity is not clearly understood. CMT is a polarized process in which the majority of the exported SPN is destined for the host cell cytosol, and is not released into the extracellular milieu. Also, CMT requires specific domains in SLO and SPN that are absolutely required for translocation, but not for pore-formation or glycohydrolase activity. We have also recently found that SLO pore-formation itself is completely dispensable for CMT. Thus, how SLO and SPN interact with the membrane and each other and how this results in polarized translocation is not clear. In addition, our recent data suggest that glycohydrolase activity may not serve as the only or most important basis of SPN's cytotoxic effect as variants of SPN that lack glycohydrolase activity are still cytotoxic and that cytotoxicity requires concomitant formation of the SLO pore. The present study will investigate the mechanism of translocation and cytotoxicity and will be furthered by our recent determination of the SPN crystal structure and our detailed kinetic analysis of SPN enzymology. Specific questions to be addressed include an analysis of a predicted glycan-binding domain in SPN, the role of SLO-membrane interactions in SPN uptake, the basis for the glycohydrolase-independent cytotoxicity and how the various activities of both SLO and SPN interact to promote toxicity and to modulate host cell signaling. Further analysis of CMT will reveal details of a novel pathway for effector translocation and how different toxins synergize to modulate host cell behavior to produce specific pathogenic outcomes. PUBLIC HEALTH RELEVANCE: Cytolysin-mediated translocation (CMT) is a novel pathway for toxin delivery in the Gram-positive pathogen Streptococcus pyogenes that uses the pore-forming cytolysin Streptolysin O (SLO) to translocate the streptococcal NAD+ glycohydrolase (SPN) across the host cell membrane and into its cytosol. Both SLO and cytosolic SPN then act to cause host cell death. In this project we will exploit our recent observations that pore-formation and glycohydrolase activity are dispensable for CMT and toxicity, respectively, along with our determination of the crystal structure of SPN to produce mutants of SPN and SLO that will be used to elucidate how SLO acts to translocate SPN. We will also use these mutants to investigate how SLO and SPN interact to both kill and manipulate the cytokine signaling responses of cells. This information will be important for understanding how the multiple toxins of S. pyogenes interact with each other and with host cells to unravel how this pathogen can cause such a diverse range of different diseases.